Traditionally, epithelial cell growth factor (EGF), fibroblast growth factor (FGF), nerve cell growth factor (NGF), platelet-derived growth factor (PDGF), endothelial cell growth factor (ECGF) and other polypeptides have been known to possess cell growth activity. In addition to those cell growth factors, a polypeptide which shows hepatocyte growth activity in vitro was partially purified from serum of rats with regenerated liver by Nakamura et al. in 1984, and named hepatocyte growth factor (hereinafter abbreviated as HGF) [Biochem. Biophys. Res. Commun., 122, 1450 (1984)].
Until the discovery of HGF, it had been impossible to cultivate hepatocytes in vitro: they showed no growth even in the presence of mammalian serum which allows vigorous growth of various lines of established cells, and they usually fell down off from the wall of cultivation vessel in about 1 week. In the presence in HGF, hepatocytes showed very good growth, and their cultivation became possible [Biochem. Biophys. Res. Commun., 122, 1450 (1984)]. Other workers confirmed that this HGF activity was present also in blood after partial hepatectomy and in blood of fulminant hepatitis patients. Although methods of purification, chemical properties and biological properties of HGF were elucidated by many workers since then, the amino acid structure of HGF or a polypeptide possessing similar hepatocyte growth activity has not been identified.
With this background, the present inventors have made a series of investigations of HGF separated and purified from various tissues such as rat platelets, and found that this platelet-derived HGF comprises two kinds of subunits and it allows hepatocytes to grow very well in vitro, and succeeded in identifying 27 amino acid residues of a partial amino acid sequence of HGF (Japanese Patent Application No. 311866/1988). Furthermore, the inventors used an oligonucleotide probe synthesized on the basis of the identified amino acid sequence to select and identify the cDNA which codes for rat hepatocyte growth factor from a rat liver cDNA library, and succeeded in selecting and identifying the cDNA which codes for human hepatocyte growth factor from a human liver cDNA library using the obtained rat cDNA [Nature, 342, 440 (1989); Japanese Patent Application No. 142697/1989]. Also, the cDNA which codes for human hepatocyt growth factor was isolated from human placenta [Biochem. Biophys. Res. Commun., 163, 967–973 (1989)] and from human leukocytes [Biochem. Biophys. Res. Commun., 172, 321 (1990)] in the same manner.
Since HGF in vivo is a polypeptide secreted in only trace amounts from organs such as liver, brain, lung, bone marrow, spleen, placenta and kidney or from blood cells such as platelets and leukocytes, there are many problems such as starting material availability. HGF yield and stable supply. To utilize this HGF for hepatocyte cultivation or hepatocyte research, it is necessary to clarify its structure and mass-produce HGF or a polypeptide possessing similar activity by gene recombination technology. Also, to clarify the relationship between diseases and HGF gene anomalies in the study of the function of HGF in vivo, it is desired to analyze the structure of the HGF gene on chromosome, which forms the basis thereof.